1. Field of the Invention
This invention relates to diagnosis of asthma and to materials and methods relating thereto.
2. Description of the Related Art
Asthma is a disease which is becoming more prevalent and is the most common disease of childhood (1). Most asthma in children and young adults is initiated by IgE mediated allergy (atopy) to inhaled allergens such as house dust mite and cat dander However, not all asthmatics are atopic, and most atopic individuals do not have asthma. Thus, factors in addition to atopy are necessary to induce the disease (2,3). Asthma is strongly familial, and is due to the interaction between genetic and environmental factors. The genetic factors are thought to be variants of normal genes (xe2x80x9cpolymorphismsxe2x80x9d) which alter their function to predispose to asthma.
Asthma may be identified by recurrent wheeze and intermittent air flow limitation. An asthmatic tendency may be quantified by the measurement of bronchial hyper-responsiveness in which an individual""s dose-response curve to a broncho-constrictor such as histamine or methacholine is constructed. The curve is commonly summarised by the dose which results in a 20% fall in air flow (PD20) or the slope of the curve between the initial air flow measurement and the last dose given (slope).
In the atopic response, IgE is produced by B-cells in response to allergen stimulation. These antibodies coat mast cells by binding to the high affinity receptor for IgE (Fcxcex5RI). When a multivalent allergen binds to an IgE-coated mast cell, the cross-linking of adjacent IgEs by allergen initiates a series of cellular events leading to the destabilisation of the cell membrane and release of inflammatory mediators. This results in mucosal inflammation, wheezing, coughing, sneezing and nasal blockage.
Atopy can be diagnosed by (i) a positive skin prick test in response to a common allergen; (ii) detecting the presence of specific. serum IgE for allergen; or (iii) by detecting elevation of total serum IgE.
Genetic associations with atopy have been demonstrated. WO 95/05481 discloses that variants of the gene encoding the xcex2-subunit of the high-affinity receptor for IgE (Fcxcex5RIxcex2) are associated with atopy. It teaches a method for diagnosing atopy which is based upon the demonstration of the presence or absence of one of two variants in a specific portion of the DNA sequence of the gene encoding Fcxcex5RIxcex2, located near the commencement of exon 6 of the Fcxcex5RIxcex2 gene on chromosome 11. A further variant has also been found in which the unusual variant sequence is in the coding sequence for the C-terminal cytoplasmic tail of Fcxcex5RIxcex2 (11).
The known polymorphisms do not account for all of the genetic factors which predispose to asthma. In particular, asthma is not necessarily an atopic disease. Identification of further genetic polymorphisms linked to asthma will allow the identification of children at risk of asthma before the disease has developed (for example immediately after birth), with the potential for prevention of disease.
Tumour necrosis factor (TNF, also known as TNFxcex1) is a potent proinflammatory cytokine that is found in increased concentration in asthmatic airways (4) and in lavage fluid from asthmatic lungs (5). Increased secretion of TNF by peripheral blood lymphocytes or monocytes has also been established in association with the HLA-DRB1*03 genotype (8). The TNF locus is located within the major histocompatibility complex (MHC) between the MHC class III genes and HLA-B. Located downstream of the TNF gene and in tandem arrangement with it is the lymphotoxin xcex1 gene (LTxcex1, which was originally designated TNFxcex2). Unlike the highly polymorphic class I and class II HLA genes, the coding portions of the TNFxcex1 and LTxcex1 genes only show a very low degree of polymorphism.
The TNFxcex1 and LTxcex1 loci have been investigated in association with autoimmune diseases such as systemic lupus erythematosus (SLE). It has been suggested that an increased level of TNF secretion is associated with allele 1 of a NcoI polymorphism in the LTxcex1 gene (9) and with allele 2 of a TNF promoter variant at position xe2x88x92308 (10). These polymorphisms are known as LTxcex1 NcoI*1 and TNF xe2x88x92308*2 respectively. However, there has been some doubt about the significance of these polymorphisms in particular, it has been suggested that the TNFxcex1 xe2x88x92308 polymorphism is not relevant to TNFxcex1 gene regulation (15, 16).
Several polymorphic microsatellite sequences within the human TNF/LT locus have also been mapped and characterised (12) and used in a cell typing study (13).
It has now been discovered that, surprisingly, unusual genetic variants in or linked to the TNFxcex1 gene are predictive of asthma Furthermore, it has been found that the unusual variants are predictive of bronchodilator and inhaled or oral steroid usage in asthmatic individuals. The variants are therefore useful to predict the clinical course of disease (e.g., severe as opposed to mild) or the response to particular treatments, as well as in a diagnostic tool. This information will be of use in relation to both individuals and population, and will be of interest to the insurance industry as well as to the healthcare and pharmaceutical industries.
These unexpected findings make possible new diagnostic and therapeutic strategies.
The present invention therefore provides a method for diagnosing an individual as being asthmatic, or of having a predisposition to asthma, which method comprises demonstrating in the individual the presence or absence of an unusual variant form of a polynucleotide sequence in the MHC region of chromosome 6p, said unusual variant form associated with an increased secretion of TNF. In particular, the variant may be located in the TNFxcex1/LTxcex1 locus, including regulatory regions for the TNFxcex1 and LTxcex1 genes. FIG. 1 shows a physical map of the human TNFxcex1/LTxcex1, locus and the locations of various polymorphisms using nomenclature as it appears in the literature The variant may be for example allele 1 of the NcoI polymorphism in the LTxcex1 gene, or it may be allele 2 of the TNF promoter polymorphism at position xe2x88x92308. Both of these variants have been described in detail previously in references 10 and 14, the contents of which are incorporated herein by reference. The method according to the invention may involve identifying the presence or absence of two or more such variants.
The sequence difference between the two alleles of the TNF xe2x88x92308 polymorphism is a single base change at position xe2x88x92308 relative to the transcription start site of the gene. For allele 1 the nucleotide at position xe2x88x92308 is a guanine xe2x80x9cGxe2x80x9d. For allele 2 the nucleotide at position xe2x88x92308 is an adenine xe2x80x9cAxe2x80x9d.
The sequence difference between the two alleles of the NcoI polymorphism in the LTxcex1 gene is a single base change at position +252 relative to the transcription start site. For allele 1 the nucleotide at position +252 is a G which forms part of a restriction site which the NcoI enzyme cuts. For allele 2 the nucleotide at position +252 is an A and the NcoI enzyme does not cut.
Suitable techniques for identifying the presence or absence of the variants are described herein. However, the invention is not limited to these specific methods. Modified versions of these methods, and alternative techniques, will be known to those skilled in the art.
A technique may be employed which uses one or more complementary nucleic acid sequences which are specific for the unusual variant and not for the wild-type. The complementary nucleic acid sequence may simply be used as a probe specific for the unusual variant. Alternatively, one or more suitable complementary nucleic acid sequences may be used as primers in a detection system comprising a polynucleotide amplification technique such as PCR or ARMS. DNA or RNA-based amplification methods may be employed. Thus the amplification may be carried out based on mRNA or cDNA made from mRNA. PCR may be followed by probing the amplification products with a sequence-specific nucleic acid probe capable of annealing to a portion of the amplified polynucleotide sequence which is specific to the unusual variant and not to the wild-type. Alternatively, confirmation of the amplification product may be obtained by probing or sequencing or by restriction digestion of the amplification product if the variant introduces or abolishes a restriction endonuclease site, or by other known methods. If the PCR primers used are specific to the variant, there will only be successful amplification if the variant is present in the test sample. Alternatively the primers used could be non-specific to the variant the amplification serving only to increase sample material for sequencing or for restriction digestion or for subsequent probing with a probe specific to the unusual variant and not to the wild-type.
Suitable specific complementary nucleic acid sequences for use as probes and primers in a detection assay for an unusual variant according to the invention, whether or not involving an amplification step, can be ascertained from the known DNA sequences. The human MHC region has been extensively characterised and sequenced. The sequence for the TNFxcex1/LTxcex1 locus is available under database reference z15026.emhum2. The nucleotide sequences for the LTxcex1 NcoI*1 and TNF xe2x88x92308*2 alleles are available in the published literature (10, 14) and are incorporated herein by reference.
The method according to the invention includes identifying variants not only by nucleic acid based techniques, but also in the case of coding sequences, by techniques which detect polymorphic forms of an amino acid sequence coded for by the nucleic acid variant. Where analysis is made of the amino acid sequence this may be done by sequencing studies, or by use of a probe which comprises an antibody binding domain which is specific for the unusual polymorphic form of the amino acid sequence. Conveniently, such probes are labelled with a detectable label.
The invention will be useful not only for the diagnosis of individuals, but also for assessing the risk of asthma across populations. Population statistics can provide organisations such as healthcare organisations and insurance companies with valuable information for planning purposes.
The invention as described may be employed together with other methods for diagnosing asthma or a predisposition to asthma. This includes other methods which detect polymorphisms, for example those described in WO 95/05481, WO 97/08338 and Hill et al (11) for diagnosing atopic asthma, or methods which investigate asthma by looking at physiological symtoms.
The invention will also be useful for predicting the clinical course of asthma, both in individuals and across populations, and the method according to the invention may therefore be performed as such. This may be used to identify asthmatic individuals who may respond to treatment directed against TNF or other pro-inflammatory molecules which interact with TNF.
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